In contrast to usually subtle mycoplasmosis, Mycoplasma alligatoris infection causes lethal multisystemic inflammatory disease of susceptible hosts. Our overall objective is to understand the roles of its secreted glycosidases, which are unprecedented among mycoplasmas, in its ecology and remarkable virulence. During other infections, exogenous ligands of bacterial origin that display pathogen-associated molecular patterns (PAMPs) stimulate host pro-inflammatory cytokine production via TLR signaling. Self-limiting responses can clear pathogens, but uncontrolled TLR activation results in exaggerated inflammation that is devastating to the host. Sterile inflammation is a response not to exogenous PAMPs but to endogenous TLR agonists liberated from damaged host tissues. Thus, ironically, ECM components degraded by bacterial glycosidases secreted during infection may mediate sterile inflammation. Infection-mediated sterile inflammation remains unexplored because of the inextricably confounding presence of intrinsic bacterial PAMPs like peptidoglycan or lipopolysaccharide. They make it difficult to partition responses to PAMPs from sterile inflammation, in order to assess their relative significance in bacterial infections. However, a mycoplasma model like M. alligatoris can avoid that confounding because it naturally lacks the relevant PAMPs, but is capable of liberating endogenous TLR agonists like degraded hyaluronan oligosaccharides (HA) from host ECM enzymatically through its glycosidase activity. We will infect primary pulmonary fibroblasts with wild type and hyaluronidase-knockout M. alligatoris to: 1) Identify TLR signaling actively elicited by infection, with focus on activation by HA and signal transduction via the key TLR adapter protein MyD88; and 2) Determine downstream effectors of TLR signaling during M. alligatoris infection, with focus on NF?B activation and TNF-? secretion as markers of the key events that mechanistically link TLR activation to sterile inflammation. The expected outcome is proof that a bacterial glycosidase can actively elicit TLR signaling involved in sterile inflammation. [unreadable] [unreadable] The work is significant because it will distinguish TLR signaling in infection-mediated sterile inflammation from responses to bacterial PAMPs. Once this becomes possible, the complete cascade of events during infection-mediated sterile inflammation in vivo can be determined much more effectively. Beyond improving our understanding of M. alligatoris' virulence, this distinction can be expected to provide a stepping stone toward evidence-based rationales for antagonists of TLRs and/or glycosidases as therapeutic interventions for management of human and animal infections caused by many species of bacteria. [unreadable] [unreadable] [unreadable] [unreadable]